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Keap1 is a BTB‐Kelch substrate adaptor protein that regulates steady‐state levels of Nrf2, a bZIP transcription factor, in response to oxidative stress. We have determined the structure of the Kelch domain of Keap1 bound to a 16‐mer peptide from Nrf2 containing a highly conserved DxETGE motif. The Nrf2 peptide contains two short antiparallel β‐strands connected by two overlapping type I β‐turns stabilized by the aspartate and threonine residues. The β‐turn region fits into a binding pocket on the top face of the Kelch domain and the glutamate residues form multiple hydrogen bonds with highly conserved residues in Keap1. Mutagenesis experiments confirmed the role of individual amino acids for binding of Nrf2 to Keap1 and for Keap1‐mediated repression of Nrf2‐dependent gene expression. Our results provide a detailed picture of how a BTB‐Kelch substrate adaptor protein binds to its cognate substrate and will enable the rational design of novel chemopreventive agents.
Eukaryote cells are constantly exposed to reactive molecules from endogenous and exogenous sources (Finkel and Holbrook, 2000; Jackson and Loeb, 2001). These molecules, including reactive oxygen species, electrophilic chemicals, and heavy metals, damage biological macromolecules and impair normal cellular functions (Davies, 2000; Imlay, 2003). Oxidative damage has been implicated in diverse pathophysiological processes, including cancer, cardiovascular disease, diabetes and neurodegeneration (Ames and Shigenaga, 1993; Jackson and Loeb, 2001; Bonnefont‐Rousselot, 2002; Ceconi et al, 2003; Ghanbari et al, 2004; Mhatre et al, 2004). Therapeutic approaches to protect against damage caused by reactive molecules would have broad ramifications for improving human health (Cash et al, 2002; Hamilton et al, 2004).
Eukaryote cells have evolved multiple protective mechanisms against reactive molecules. A major mechanism is the coordinated induction of enzymes that neutralize reactive molecules, eliminate damaged macromolecules and restore cellular redox homeostasis. In metazoan organisms, this cytoprotective response is controlled, in large part, by the Cap N' Collar (CNC) transcription factors, which constitute a unique subset within the bZIP transcription factor family (Mathers et al, 2004). In mammals, the CNC transcription factors include Nrf1, Nrf2, Nrf3, Bach1 and Bach2 (Motohashi et al, 2002). Phenotypic analysis of mice containing targeted deletions of individual or multiple CNC family members has revealed that Nrf1 and Nrf2 are the major regulators of cytoprotective gene expression (Leung et al, 2003; Yu and Kensler, 2005). Nrf1 and Nrf2 share a common domain structure, including a conserved N‐terminal regulatory domain termed Neh2, a central transactivation domain, and a C‐terminal bZIP domain required for heterodimer formation with members of the small Maf protein family, for DNA binding and nuclear import and export (Chan et al, 1993; Moi et al, 1994; Katoh et al, 2001; Bloom et al, 2002; Motohashi et al, 2002; Li et al, 2005).
The Keap1 protein is the major negative regulator of cytoprotective gene expression (Itoh et al, 1999; Dhakshinamoorthy and Jaiswal, 2001). Keap1, a BTB‐Kelch protein, is a substrate adaptor protein for a Cul3‐dependent ubiquitin ligase complex (Cullinan et al, 2004; Kobayashi et al, 2004; Zhang et al, 2004). Under basal conditions, Keap1 targets Nrf2 for ubiquitin‐dependent degradation and represses Nrf2‐dependent gene expression (Wakabayashi et al, 2003; Zhang and Hannink, 2003). In cells exposed to reactive chemicals or oxidative stress, Nrf2 is no longer targeted for ubiquitin‐dependent degradation (Zhang and Hannink, 2003; Zhang et al, 2004; Kobayashi et al, 2006). Instead, steady‐state levels of Nrf2 increase, resulting in activation of Nrf2‐dependent gene expression.
Keap1, as a substrate adaptor, bridges both Cul3 and Nrf2 using its N‐terminal BTB and central linker domains to bind Cul3 and its C‐terminal Kelch domain to bind the Neh2 domain of Nrf2 (Cullinan et al, 2004; Kobayashi et al, 2004; Zhang et al, 2004). Lysine residues within the Neh2 domain of Nrf2 are targeted for ubiquitin transfer mediated by the Cul3‐associated Rbx1 protein and a ubiquitin‐charged E2 protein (Zhang et al, 2004). Cyclical association and dissociation of this E3 ubiquitin ligase complex, mediated by the opposing actions of CAND1 and Cul3 neddylation, enables efficient ubiquitination of Nrf2 and repression of Nrf2‐dependent gene expression (Lo and Hannink, 2006). This ubiquitin ligase complex is perturbed by reactive chemicals and oxidative stress, which modify a number of reactive cysteine residues located in the BTB and linker domains of Keap1 (Dinkova‐Kostova et al, 2002; Zhang and Hannink, 2003; Wakabayashi et al, 2004; Eggler et al, 2005; Hong et al, 2005a, 2005b).
We have determined the structure of the Kelch domain of Keap1 bound to a peptide containing a conserved DxETGE motif located in the Neh2 domain of Nrf2. The binding pocket in Keap1 contains multiple charged and hydrophobic residues that contact the side chains of the two glutamate residues and the peptide backbone. The DxETGE motif contains a β‐turn region stabilized by the aspartate and threonine residues. Threonine phosphorylation prevents binding of the Nrf2‐derived peptide to Keap1 and phosphomimetic mutations enable Nrf2 to escape Keap1‐mediated repression. Keap1 dimerizes via its BTB domain and both Kelch domains within the Keap1 dimer are capable of independently binding to Nrf2. Our results provide insight into how Keap1 targets Nrf2 for ubiquitin‐dependent degradation and will facilitate the rational design of novel therapeutic compounds that block binding of Nrf2 to Keap1.
The structure of a complex between the Kelch domain of Keap1 and an Nrf2‐derived peptide reveals how Nrf2 fits into the substrate binding pocket of Keap1
The Kelch domain of Keap1 is a six‐bladed β‐propeller in which each blade of the propeller (I–VI) is comprised of four β‐strands (A–D) (Figure 1A) (Li et al, 2004). The β‐strands are connected by loops of varying lengths that project from the central core of the β‐propeller. By convention (Wall et al, 1995), the short loops that connect either β‐strands A and B (A–B) or β‐strands C and D (C–D) define the bottom face of the β‐propeller, whereas the longer loops that connect either β‐strands D and A (D–A) or β‐strands B and C (B–C) define the top face of the β‐propeller.
The Neh2 domain contains two regions that are highly conserved from humans to flies (Figure 1B). One region, amino acids 17–32 in the human Nrf2 protein, contains an LxxQDxDLG motif (McMahon et al, 2004; Katoh et al, 2005). A second region, amino acids 77–82 in the human Nrf2 protein, contains a DxETGE motif (Kobayashi et al, 2002). Both of these motifs within Nrf2 bind to the same site in Keap1 but the affinity of Keap1 for the DxETGE motif is approximately 100‐fold higher than for the LxxQDxDLG motif (Tong et al, 2006).
To determine if the high‐affinity DxETGE motif is sufficient for binding to Keap1, the ability of several different DxETGE‐containing peptides to disrupt the Keap1–Nrf2 complex was determined. A complex between a Keap1 protein containing a C‐terminal chitin binding domain (Keap1‐CBD) and an HA‐tagged Nrf2 protein was isolated by affinity purification on chitin beads. The beads were incubated with increasing amounts of the peptides and levels of bound HA‐Nrf2 determined. A marked reduction in the amount of HA‐Nrf2 that remained bound to the Keap1‐CBD protein was observed following incubation with a 16‐mer Nrf2‐derived peptide containing amino acids 69–84 (AFFAQLQLDEETGEFL) (Figure 2A, lanes 11–14). A 14‐mer peptide, containing amino acids 74–87 of Nrf2 (LQLDEETGEFLPIQ), was able to displace Nrf2 from Keap1 as effectively as the 16‐mer (Figure 2A, lanes 7–10). A 10‐mer Nrf2‐derived peptide, containing amino acids 76–85 (LDEETGEFLP), was also able to displace Nrf2 from Keap1, although not quite as effectively as the two longer peptides (Figure 2A, lanes 3–6). This 10‐mer peptide lacks one or more backbone interactions that stabilize the longer peptides (see Figure 4C). Isothermal titration calorimetry revealed that the 16‐mer peptide bound to the Kelch domain of Keap1 with a Kd value of 20 nM (Figure 2B), in good agreement with the published Kd values ranging from 5 to 9 nM for binding of the Neh2 domain to the full‐length Keap1 protein (Eggler et al, 2005; Tong et al, 2006).
The structure of the human Kelch domain bound to the 16‐mer peptide was determined to be 1.5 Å using X‐ray crystallography (Figure 3). As predicted by our previous work (Li et al, 2004), and in agreement with the recent structural analysis of the mouse Kelch domain bound to a DxETGE‐containing peptide (Padmanabhan et al, 2006), the Nrf2‐derived peptide binds in the shallow pocket defined by the D–A and B–C loops on the top face of the Kelch domain (Figure 3). The peptide has two antiparallel β‐strands connected by a turn region that has two overlapping type I β‐turns (residues 77–80 and 78–81; Figure 4C). The turn region is stabilized by hydrogen bonds involving the side chains of D77 and T80 and the peptide backbone (Figure 4C). Complex formation buries 420 Å2 of the surface area on the Kelch β‐propeller.
Glutamate residues E79 and E82 are the only peptide residues whose side chains make specific interactions with the Kelch domain (Figure 4A and Table I). The carboxylate oxygen atoms of E79 contact the side chains of Arg415, Arg483, and Ser508, whereas the carboxylate oxygen atoms of E82 make hydrogen bonds with the side chains of Ser363, Asn382, and Arg380. The peptide backbone makes five contacts with the Kelch domain, four from the carbonyl oxygen atoms of E78, E79, T80, and F83, and one from a backbone amide group of F83 (Figure 4B).
Solvent molecules mediate additional contacts between the peptide and the Kelch domain (Table II), enabling an additional contact between E79 and Arg415 and allowing E82 to interact with both Asn414 and Ser602. The carboxylate oxygen atom of D77 contacts two water molecules that make bridging hydrogen bonds with Arg415 and Arg380. The hydroxyl group of T80 utilizes a water molecule to interact with Arg380.
All six blades of the Kelch β‐propeller contribute to complex formation (Table I). Kelch domain residues that contact the side chains of the peptide are concentrated on one side of the binding pocket in blades II, III, IV, and V, whereas residues that contact backbone atoms of the peptide or participate in van der Waals interactions are located on the other side of the binding pocket in blades V, VI, I, and II (Tables I and II).
Side chains from six residues in Keap1 (Ser363, Asn382, Arg380, Arg415, Arg483, and Ser508) participate in hydrogen bond interactions with the carboxylate oxygen atoms from E79 and E82 in the peptide (Figure 4A). Some of these six residues also participate in other interactions with the peptide. For instance, the backbone amide of Asn382 interacts with the carbonyl oxygen of F83, and the side chains of both Arg415 and Arg380 form a hydrogen bond to a solvent water molecule that bridges to the carboxylate oxygen of D77 (Table II). Side chains from five additional residues in Keap1 participate in hydrogen bonding with the peptide backbone: these are Tyr334, Asn382, Gln530, Ser555, and Ser602 (Figure 4B). Finally, the side chains from seven residues in Keap1 participate in van der Waals interactions with the peptide, including Tyr334, Asn387, Arg415, Ser508, Tyr525, Tyr572, and Phe577 (Table I).
Identification of amino acids within the binding pocket of Keap1 that are required for binding to Nrf2
An extensive alanine‐scan mutagenesis of Keap1 was performed to determine the contribution of specific amino acids in Keap1 for binding of Nrf2. Twelve Keap1 mutants that contained alanine substitutions for three or four adjacent residues within each B–C loop were constructed (Table I). The ability of the mutant Keap1 proteins to bind Nrf2 was determined by co‐immunoprecipitation and their ability to repress Nrf2‐dependent transcription was determined by a reporter gene assay using an ARE‐dependent luciferase gene. Eight of the 12 mutant Keap1 proteins were impaired in their ability to bind to Nrf2 and to repress Nrf2‐dependent gene transcription (Figure 5A and B).
The alanine‐scan mutagenesis was refined in a second set of mutant Keap1 proteins, in which 13 amino acids in the B–C loops, each with a surface‐exposed side chain, were individually mutated to alanine (Table I). Substitution of individual alanine residues for Tyr334, Asn382, His436, Tyr525, and Tyr572 significantly disrupted the ability of Keap1 to bind Nrf2 and repress Nrf2‐dependent transcription (Figure 5C and D). With the exception of His436, all of these residues were observed in the crystal structure to participate in contacts with the Nrf2‐derived peptide (Figure 4 and Tables I and II). It is not clear why alanine substitution at His436 perturbed the ability of Keap1 to bind Nrf2, as the circular dichroic (CD) spectrum of the His436A Kelch domain is nearly identical to the CD spectrum of the wild‐type Kelch domain (Supplementary Figures 1 and 2). An additional residue, Phe478, was not required for binding to Nrf2 (Figure 5C), but was required for repression of Nrf2‐dependent gene expression (Figure 5D). Further analysis revealed that this mutant was defective for directing ubiquitination onto Nrf2 (data not shown).
A third set of mutant Keap1 proteins were constructed in which six arginine residues and one lysine residue, all with surface‐exposed side chains, were substituted with alanine residues (Table I). This analysis identified three arginine residues (Arg380, Arg415, Arg483) that were required for binding to Nrf2 and for repression of Nrf2‐dependent gene expression (Figure 5E and F). As seen in the crystal structure, the side chains of these three arginine residues are involved in multiple contacts with the Nrf2‐derived peptide (Figure 4 and Tables I and II).
A final set of mutant Keap1 proteins were constructed that contained alanine residues individually substituted for four serine residues located in the D–A loops, Ser363, Ser508, Ser555, and Ser602 (Table I). Although the side chains of these serine residues are involved in hydrogen bond interactions with the Nrf2‐derived peptide (Figure 4), individual alanine substitutions of these serine residues did not significantly reduce the ability of Keap1 to bind to Nrf2 or to repress Nrf2‐dependent gene expression (Figure 5G and H).
Keap1 forms a homodimer capable of binding two Nrf2 molecules
The structure of the human Kelch domain bound to a 16‐mer Nrf2‐derived peptide and the recently published structure of the murine Kelch domain bound to a shorter Nrf2‐derived peptide (Padmanabhan et al, 2006) indicate that the Kelch domain of Keap1 contains a single binding site for the DxETGE motif in Nrf2. As Keap1 dimerizes via its N‐terminal BTB domain (Zipper and Mulcahy, 2002), the structural data are consistent with the notion that Keap1 and Nrf2 form a complex with a 2:2 stoichiometry. However, several recent reports have suggested that the Keap1 dimer binds one Nrf2 polypeptide, resulting in a 2:1 stoichiometry (Eggler et al, 2005; Tong et al, 2006). As these in vitro biochemical experiments used high concentrations of purified proteins obtained from prokaryote expression systems, the stoichiometry of the Keap1:Nrf2 complex in the more complex environment of a eukaryote cell remains an open question.
To confirm that Keap1 exists as a dimer in eukaryote cells, a CBD‐tagged Keap1 protein was coexpressed in HEK 293 T cells along with an untagged Keap1 protein. The untagged Keap1 protein was pulled down with chitin beads only in the presence of CBD‐tagged Keap1 (Figure 6A, second panel from top, compare lane 13 with lane 14). This result is consistent with a prior report that protein–protein interactions involving the N‐terminal BTB domain enable Keap1 to form a homodimer (Zipper and Mulcahy, 2002), although the possibility that Keap1 forms higher order oligomeric structures in cells cannot be ruled out. As expected, Nrf2 was pulled down with chitin beads in the presence of the CBD‐tagged wild‐type Keap1 protein (Figure 6A, top panel, lanes 11 and 12).
Next, we generated heterodimeric Keap1 complexes containing one wild‐type Kelch domain and one mutant Kelch domain to determine if the presence of a single functional Kelch domain in a Keap1 dimer would be sufficient to bind Nrf2. A CBD tag was appended to the mutant Keap1 protein such that two types of Keap1 dimers would be isolated on chitin beads: a Keap1 homodimer containing two mutant Kelch domains and a Keap1 heterodimer containing one wild‐type and one mutant Kelch domain. Three mutant Keap1 proteins were used for this analysis, including two that were unable to bind Nrf2 on their own (Y334A and R415A) and one (Q337A) that was able to bind Nrf2 as well as the wild‐type Keap1 protein. The ability of the homodimeric and heterodimeric complexes to bind Nrf2 was assessed by chitin pulldown followed by immunoblot analysis. As expected, no Nrf2 was pulled down with chitin beads in the presence of either the Keap1‐Y334A and Keap1‐R415A proteins (Figure 6A, upper panel, lanes 2 and 8). However, coexpression of the wild‐type untagged Keap1 protein with either of these two mutant Keap1 proteins partially restored pulldown of Nrf2 (Figure 6A, upper panel, compare lanes 3, 9, and 12). Partial restoration of Nrf2 pulldown is expected, as only a portion of the dimeric Keap1 complexes isolated on chitin beads would have one wild‐type Kelch domain with the remainder of the Keap1 complexes containing two non‐functional Kelch domains. These results demonstrate that a single functional Kelch domain within the context of a Keap1 dimer is sufficient to bind Nrf2.
Finally, we asked if the Keap1 dimer is capable of binding two different Nrf2 proteins. The Neh2 domain of Nrf2 was fused onto the Gal4 DNA binding domain and coexpressed with HA‐tagged full‐length Nrf2 protein in the absence or presence of coexpressed Keap1. Cell lysates were immunoprecipitated with anti‐HA antibodies and the resultant immunoprecipitates were probed for the presence of the Gal4‐Neh2 protein using anti‐Gal4 antibodies. The Gal4‐Neh2 protein was only present in the anti‐HA immunoprecipitates in the presence of coexpressed Keap1 (Figure 6B, upper panel, compare lanes 2 and 3). Thus, the Keap1 protein functions as a bridge between the HA‐Nrf2 protein and the Gal4‐Neh2 protein. A single alanine substitution for T80 within the DxETGE motif of HA‐Nrf2 abolished co‐immunoprecipitation of the Gal4‐Neh2 protein (Figure 6B, upper panel, lane 4). The Keap1‐Y334A protein, which is not able to bind either HA‐Nrf2 or Gal4‐Neh2, is not able to bridge the two Nrf2 proteins (Figure 6B, upper panel, lane 5). Likewise, the isolated Kelch domain, which is not able to form a homodimer, is not able to bridge the two Nrf2 proteins although it readily binds to the HA‐Nrf2 protein (Figure 6B, upper panel, lane 6). These results indicate that a Keap1 dimer is able to bridge two different DxETGE‐containing proteins, consistent with the notion that Keap1 and Nrf2 can form a 2:2 stoichiometric complex.
Phosphorylation of the conserved threonine within the DxETGE motif in Nrf2 disrupts binding to Keap1
The crystal structure of the Nrf2‐derived peptide bound to the Kelch domain revealed the presence of two overlapping type I β‐turns, residues 77–80 and 78–81, in which the side chains of D77 and T80, respectively, form hydrogen bonds with the peptide backbone. The threonine residue T80 was of particular interest, given the possibility that phosphorylation of this residue might interfere with formation of the β‐turn and prevent binding of Nrf2 to Keap1.
Peptide competition experiments were carried out using 14‐mer peptides that contained either an alanine or phosphothreonine in place of T80. A 14‐mer peptide corresponding to amino acids 74–87 of Nrf2 that contained an alanine in place of T80 was unable to displace HA‐Nrf2 from Keap1‐CBD even at input peptide levels 100‐fold higher than that needed for the wild‐type peptide to displace Nrf2 from Keap1 (Figure 7A, compare lanes 3–6 with lanes 7–10). Likewise, a 14‐mer peptide that contained a phosphothreonine residue in place of T80 was also unable to displace Nrf2 from Keap1 (Figure 7A, lanes 11–14).
To examine the importance of T80 for Keap1‐mediated repression of Nrf2‐dependent gene expression in cells, we characterized several mutant Nrf2 proteins in which T80 was altered to alanine, serine, or to the phosphomimetic glutamate or aspartate. Consistent with the peptide competition experiments, the T80A Nrf2 mutant protein did not bind Keap1 and was resistant to Keap1‐mediated repression of Nrf2‐dependent gene expression (Figure 7B, top panel, lane 2, and Figure 7C). Nrf2 proteins containing either of the phosphomimetic substitutions (T80D and T80E) were also impaired in binding to Keap1 and were not subject to Keap1‐mediated repression (Figure 7B, top panel, lanes 4 and 5, and Figure 7C). In contrast, the T80S mutant Nrf2 protein behaved essentially like the wild‐type Nrf2 protein, with only a slight reduction of binding of Nrf2 to Keap1 and Keap1‐mediated repression (Figure 7B, top panel, lane 3, and Figure 7C). These results indicate that the hydroxyl group of the serine residue is an effective substitute for the hydroxyl group of the threonine residue in stabilizing the β‐turn of the DxETGE motif. In contrast, either the absence of the threonine hydroxyl group or the presence of a negative charge disrupts the β‐turn region and likely prevents the flanking glutamate residues from interacting with their cognate residues in Keap1.
The ability of cullin‐dependent ubiquitin ligase complexes to carry out protein ubiquitination in a highly specific and regulated manner derives from the ability of substrate adaptor proteins to recognize their substrates with high affinity and specificity. The substrate adaptor proteins for the Cul1 and Cul2 proteins have been intensively studied and structural analyses have provided a detailed understanding of their ability to recognize specific substrates (Hon et al, 2002; Min et al, 2002; Orlicky et al, 2003; Wu et al, 2003). Recent work has suggested that the BTB‐Kelch proteins, defined by an N‐terminal BTB domain and a C‐terminal Kelch domain, function as substrate adaptor proteins for Cul3 (Cullinan et al, 2004; Kobayashi et al, 2004, 2006; Zhang et al, 2004, 2005; Furukawa and Xiong, 2005; Wang et al, 2005). In this report, we have determined the structure of the Kelch domain of human Keap1 bound to a 16‐mer peptide derived from its substrate, Nrf2. Together with a structure of the Kelch domain of murine Keap1 bound to a 9‐mer Nrf2‐derived peptide published while this manuscript was under review (Padmanabhan et al, 2006), these structural studies provide a detailed picture of how Keap1 binds Nrf2.
Comparison of amino‐acid contacts between the Kelch domain and the Nrf2‐derived peptide with functional analysis of mutant Keap1 proteins containing individual alanine substitutions indicates that charged residues and hydrophobic residues located in the binding pocket of Keap1 are the major contributors to the stability of the complex. In particular, the side chains of Tyr334, Arg380, Asn382, Arg415, Arg483, Tyr525, and Tyr572 in Keap1 each contact the Nrf2‐derived peptide and individual alanine substitutions for these residues significantly impair binding to Nrf2 and repression of Nrf2‐dependent gene expression. In contrast, individual alanine substitutions of four serine residues (Ser363, Ser508, Ser555, and Ser602) that directly contact the Nrf2‐derived peptide did not significantly impair binding of Nrf2 or repression of Nrf2‐dependent transcription. Thus, hydrogen bond contacts between these serine residues and Nrf2 do not contribute significantly to the overall stability of the Keap1:Nrf2 complex. Nevertheless, the overall agreement between our structural and functional studies indicates that the crystal structure of the complex between the Kelch domain of Keap1 and the Nrf2‐derived peptide is an accurate representation of amino‐acid contacts between the full‐length Keap1 and Nrf2 proteins.
Our structural and functional studies provide strong support for the notion that the DxETGE motif is the principal Keap1 binding site in Nrf2. The lysine residues in Nrf2 that are targeted for ubiquitination are located at a distance of 10–30 amino acids on the N‐terminal side of the DxETGE motif (Zhang et al, 2004). Binding of Nrf2 to Keap1 via the DxETGE motif would position these residues for ubiquitin transfer. A second Keap1 binding site in Nrf2, containing the consensus sequence LxxQDxDLG and located at a distance of approximately 50 amino acids on the N‐terminal side of the DxETGE motif, has recently been identified (Tong et al, 2006). The LxxQDxDLG motif binds in the substrate binding pocket of the Kelch domain although the affinity of Keap1 for the LxxQDxDLG motif is approximately two orders of magnitude less than for the DxETGE motif. Yamamoto and co‐workers (Tong et al, 2006) have proposed a model in which one Nrf2 polypeptide, via both the DxETGE and the LxxQDxDLG motifs, bridges the two Kelch domains in the Keap1 homodimer. This model is supported by in vitro biochemical experiments using purified proteins that suggest a 2:1 stoichiometry of the Keap:Nrf2 complex (Eggler et al, 2005; Tong et al, 2006). In contrast, our experiments, performed by ectopic expression of Keap1 and Nrf2 proteins in the complex intracellular milieu of a eukaryote cell, indicate that the two Kelch domains of a Keap1 homodimer are capable of binding independently to two different Nrf2 polypeptides, consistent with a 2:2 stoichiometry for the Keap:Nrf2 complex. A structure‐based model of the Keap1:Cul3:Rbx1 complex suggests that the two Kelch domains in the Keap1 dimer are spatially separated (Stogios et al, 2005), consistent with the notion that each Kelch domain can independently present a substrate protein to the E2 conjugating enzyme recruited into the E3 ubiquitin ligase complex by Rbx1. As this structural model is largely based on computational modeling, detailed structural and functional analysis of the entire ubiquitin ligase complex is needed to provide further insight into how Keap1 presents Nrf2 as a substrate for ubiquitination.
Keap1 constitutively targets Nrf2 for ubiquitin‐dependent degradation under basal conditions of cell growth. Following exposure of cells to electrophilic chemicals or overt oxidative stress, Nrf2 is able to escape Keap1‐mediated degradation, accumulate in the nucleus, and activate gene expression. The prevailing model for activation of Nrf2 is that reactive chemicals and oxidative stress modify one or more cysteine residues in Keap1 that are located in the BTB and linker domains (Dinkova‐Kostova et al, 2002; Zhang and Hannink, 2003; Wakabayashi et al, 2004; Eggler et al, 2005; Hong et al, 2005a, 2005b). As a result, the ability of Keap1 to assemble into a functional E3 ubiquitin ligase complex with Cul3 is perturbed and Nrf2 is no longer efficiently targeted for ubiquitin‐dependent degradation although Nrf2 remains associated with Keap1 (Zhang and Hannink, 2003; Zhang et al, 2004; Kobayashi et al, 2006). However, our present results indicate that phosphorylation of threonine residue T80 within the DxETGE motif markedly decreases the ability of an Nrf2‐derived peptide to compete with Nrf2 for binding to Keap1 in vitro. Furthermore, phosphomimetic mutants of Nrf2 escape Keap1‐mediated repression in cells. As the side chain of T80 participates in hydrogen bonds that stabilize the β‐turn, which, in turn, enables the DxETGE motif to fit into the binding pocket of Keap1, phosphorylation of T80 is likely to perturb the conformation of the peptide so that it is unable to fit into the binding site on Keap1. The bulky phosphoryl group may also provide steric hindrance. Although phosphorylation of T80 has not been demonstrated in vivo, an intriguing hypothesis is that phosphorylation of T80 may enable activation of Nrf2 in the absence of overt exposure to electrophilic compounds or oxidative stress. A detailed investigation of the phosphorylation status of T80 in Nrf2 in vivo will provide novel insights into the multiplicity of pathways that can lead to activation of Nrf2‐dependent gene expression.
Keap1 is one of 49 BTB‐Kelch proteins encoded by the human genome. Point mutations in the Kelch domain of several BTB‐Kelch proteins, including Keap1, gigaxonin, and ENC1, have been associated with human diseases (Figure 1A). In the case of Keap1, one gene variant (G364C) and one somatic mutation (G430C) have been associated with lung cancer (Padmanabhan et al, 2006). The G364C mutation is located in the A–D loop of blade I and likely disrupts the substrate binding pocket, whereas the G430C mutation is adjacent to the B strand in blade III and likely disrupts the structure of the Kelch domain. As expected, both of these mutant Keap1 proteins are impaired in their ability to bind Nrf2 (Padmanabhan et al, 2006). A number of disease‐associated point mutations within the Kelch domains of both gigaxonin and ENC1 have been described (Bomont et al, 2000, 2003; Kuhlenbaumer et al, 2002; Liang et al, 2004) and it is likely that these mutations will perturb the association of gigaxonin and ENC1 with their respective substrate proteins. Furthermore, as the overall dimension of the substrate binding pocket, formed by the D–A and the B–C loops, is likely to be very similar in other BTB‐Kelch proteins, substrate(s) for other BTB‐Kelch proteins may also utilize β‐turn motifs to bind their cognate substrate adaptor. The Keap1 protein has emerged as a representative prototype for the BTB‐Kelch proteins and a detailed understanding of the structure and function of Keap1 will continue to illuminate our understanding of this large and important family of human proteins.
Materials and methods
Recombinant DNA molecules and peptides
Eukaryote expression plasmids for the wild‐type Keap1, CBD‐tagged Keap1, HA‐tagged Nrf2 and Gal4‐Neh2 proteins, and the ARE‐dependent reporter plasmid have been described previously (Zhang et al, 2004). The pET15b‐based expression vector for the His‐tagged Kelch domain of Keap1 has been described previously. Mutant Keap1 and Nrf2 cDNAs were generated by site‐specific mutagenesis with standard overlap‐extension techniques. All the genes used in this study were sequenced in the context of the expression vectors used for the experiments. Peptides were purchased from Sigma‐Genosys and from Cell‐tek and were supplied at greater than 95% purity following HPLC purification.
Cell culture, transfections, and reporter gene assays
COS1, MDA‐MB‐231, and HEK‐293 T cells were purchased from ATCC. Cells were maintained in either Dulbecco's modified Eagle's medium or Eagle's minimal essential medium in the presence of 10% fetal bovine serum. Transfections were performed with Lipofectamine Plus (Gibco BRL). Reporter gene assays were performed using the Promega Dual‐Light assay system.
Antibodies, immunoprecipitation, peptide competition, and immunoblot analyses
The anti‐Keap1 antibody has been described (Zhang and Hannink, 2003). Antibodies against Nrf2 (Santa Cruz), chitin binding domain (New England Biolabs), and the HA epitope (Covance) were purchased from commercial sources. For immunoprecipitation, cell extracts were prepared in RIPA buffer (10 mM sodium phosphate (pH 7.9), 150 mM NaCl, 1% Triton X‐100, 1% sodium deoxycholate, 0.1% Triton SDS) containing 1 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride, and protease inhibitor cocktail (Sigma). Soluble cell lysates were incubated with 2 μg of affinity‐purified antibodies for 2 h at 4°C followed by incubation at 4°C with protein A‐agarose beads (Sigma) for 2 h. Unbound proteins were removed by washing four times with lysis buffer. The immunoprecipitated proteins were eluted in sample buffer by boiling for 5 min, electrophoresed through SDS–polyacrylamide gels, transferred to nitrocellulose membranes, and subjected to immunoblot analysis.
For peptide competition assay, COS1 cells were transfected with expression vectors for HA‐Nrf2 and Keap1‐CBD. The transfected cells were exposed to proteasome inhibitor MG132 for 4.5 h before cell lysis. Cell lysates were collected in ELB buffer (50 mM Hepes (pH 7.9), 250 mM NaCl, 0.1% Triton X‐100, 5 mM EDTA) containing 1 mM DTT, 1 mM phenylmethylsulfonyl fluoride, protease inhibitor cocktail, and phosphatase inhibitors (1 mM EGTA, 1 mM Na3VO4, 100 mM NaF, 20 mM sodium pyrophosphate). The lysates (200 μg) were incubated with chitin bead at 4°C, pelleted by centrifugation (3000 g), and washed in ELB buffer. Proteins that remained associated with the chitin beads were further incubated with the peptides (10 ng, 100 ng, 1 μg, or 10 μg) at 4°C and, after several washes in ELB buffer, were analyzed by immunoblotting with anti‐HA and anti‐CBD antibodies.
Isothermal titration calorimetry
Isothermal titration calorimetry experiments were conducted at 25°C in a MicroCal VP‐ITC. The Kelch protein was dialyzed to equilibrium against the reaction buffer (50 mM Tris, pH 8.0, 100 mM NaCl, 5 mM DTT). An aliquot of the dialysis reservoir was retained for preparing dilutions of the Kelch domain and peptide. Immediately before the experiment, the protein and peptide solutions were briefly degassed under vacuum. Additions (10 μl) of the peptide were made, at 4 min intervals, to samples of the Kelch domain contained in the 1.41 ml sample cell. In three experiments, a 5 μM solution of the Kelch domain was titrated with 50 μM peptide. In a fourth experiment, a 6.25 μM solution of the Kelch domain was titrated with 88 μM peptide. The integrated data from the four experiments were analyzed globally, employing a singe‐site binding model, to extract estimates for the binding constant and binding enthalpy.
Crystallization and data collection
Purification of the Kelch domain was carried out as previously described (Li et al, 2004). Crystals were grown by hanging drop vapor diffusion at 4°C from 2 μl of a 1:3 molar ratio mixture of protein to peptide and 2 μl of well buffer. Diffraction quality crystals grew within 2 days from a well buffer containing 0.2 M MgCl2, 0.1 M Tris–HCl, and 30% w/v PEG 4000, at a protein concentration of 10 mg/ml. A single crystal was used to collect a 1.55 Å data set at −180°C on beamline MBC 4.2.2 at the Advanced Light Source of Lawrence Berkeley National Laboratory. All data were indexed and integrated with d*Trek (Pflugrath, 1999). Data collection and refinement statistics are given in Table III.
Structure solution and refinement
The structure of Kelch‐peptide complex was determined by molecular replacement using MOLREP (Murshudov et al, 1997) and the structure of the Kelch domain (1U6D) as the search model. Refinement was performed with REFMAC 5.0 (Murshudov et al, 1999); progress was monitored by use of Rfree and 5% of the data were set aside for crossvalidation before refinement. Water molecules were placed in peaks greater than 3.0σ in Fo−Fc maps and within hydrogen bonding distance to nitrogen or oxygen atoms of the protein, or other solvent atoms. TLS refinement (Winn et al, 2001) was performed with the protein and peptide as separate rigid bodies. Model building was performed interactively using Coot (Emsley and Cowtan, 2004).
The final model of the protein consisted of 285 residues in the Kelch domain, 16 residues in the peptide, and 333 water molecules (Table III). Residues are numbered according to the sequence of the intact Keap1 protein and Nrf2 proteins. Clear electron density was present for the backbone of all residues of the peptide; residues at the termini of the peptide are involved in crystal contacts with other molecules in the unit cell. Density for the side chains of five residues in the Kelch domain (Glu446, Arg447, Glu493, Gln528, and Lys551) and two in the peptide (T72 and E78) was not well defined and is represented as alanine residues. A total of 17 residues have been modeled in two conformations, including one in the peptide (Q75). The model was evaluated by SFCHECK (Vaguine et al, 1999) and WHAT_CHECK (Hooft et al, 1996). Figures were prepared with PYMOL (De Lano, 2002). The coordinates have been deposited in the PDB with code 2FLU. The model has good geometry, with 91.6% of its residues in the most favored regions of the Ramachandran plot and 0.0% in the disallowed regions (Laskowski et al, 1993).
Supplementary data
Supplementary data are available at The EMBO Journal Online.
Supplementary Information
Supplementary Figure 1 [emboj7601243-sup-0001.tiff]
Supplementary Figure 2 [emboj7601243-sup-0002.tiff]
We are indebted to Dr Jay Nix and the staff of beamline 4.2.2 at the Advanced Light Source of Lawrence Berkeley Laboratory for collection of the synchrotron data. We thank Dr Jack Tanner and Dr Shrikesh Sachdev for helpful discussions. This work was supported by a research grant from NIH (AT003899) and a development project in P50 CA103130.
- Copyright © 2006 European Molecular Biology Organization


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The Lightning Fart: A Parody of The Lightning Thief
- Percy Jackson & the Olympians, Book 1
- Lu par : Brian Holden
- Durée : 2 h et 55 min
- Version intégrale Livre audio
- Date de publication : 22/12/2017
- Langue : Anglais
- Éditeur : Steve Lookner
Prix : 6,24 €
Meet Percy Stinkson. He's just a normal kid, trying to have fun and make it through school doing as little homework as possible....
until he finds out he's the son of a Greek god! And Percy's father isn't just any Greek god. He's Pooseidon, God of Farts!
Percy's life is totally transformed as he travels to Camp Half-Wit, where he's trained to use his special farting powers. He's going to need these powers, because he soon learns it's up to him to save the universe by going on a quest to find Zeus's stolen iPhone!
It'll be a dangerous quest, but Percy will have company: Grover, a weird kid who thinks he's a half-goat, and Annabeth, a super-hot girl who unfortunately doesn't want to date Percy. Yet.
There'll be loads of action and tons of smelly farts as Percy's quest proceeds to its exciting conclusion: a showdown in the Underworld with Hades himself!
The odds might be against Percy, but don't give up hope, because he'll have the ultimate weapon at his disposal: The Lightning Fart!
Can a fart really save the universe?
Find out...in this hilarious parody of the Percy Jackson books, THE LIGHTNING FART!
Steve Lookner is one of Amazon's top parody authors. He began his writing career as an editor of The Harvard Lampoon, and has written for TV shows such as Saturday Night Live and MADtv. Be sure to check out Steve's other parodies on Amazon, including The Maze Bummer: A Parody of the Maze Runner, and City of Boneheads: A Parody of City of Bones (The Mortal Instruments, Book 1).
Autres livres audio du même :
Ce que les membres d'Audible en pensent
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- Elisabeth H
- 27/12/2017
An Excellent Parody that's clean enough for kids
Where does The Lightning Fart: A Parody of The Lightning Thief rank among all the audiobooks you've listened to so far?
This book is very different from what I usually listen to, so it's hard to compare. The reader is better than most--obviously a trained actor with lots of expression.
What other book might you compare The Lightning Fart: A Parody of The Lightning Thief to and why?
In the tradition of Bored of the Rings, this Percy Jackson parody had me laughing along all the way through. I was surprised how closely it followed the original book. So if you're a fan of the series, all your favorite scenes and characters are here.
What about Brian Holden's performance did you like?
Who better to read a Percy Jackson parody than Brian Holden of Team StarKid fame (A Very Potter Musical)? Chicago comedian Holden strikes all the right notes and provides brilliant voices for the distinct characters. I felt like I was watching this book happen in front of me rather than just listening to someone read! His inflection and tone matched the book perfectly. I definitely want to hear him read more!
Was there a moment in the book that particularly moved you?
It's not so much that I was moved, as that something moved within me.
Any additional comments?
I was very impressed that the author kept this clean enough for the kids who read Percy Jackson. Even though it's all about farts, it never goes over the top into really gross territory. My 11-year-old will definitely enjoy it.
It's just so funny! If you love Percy Jackson, you have got to hear this!
2 sur 2 personne(s) ont trouvé cet avis utile.
- SidneyAnne Hamelin
- 01/04/2018
A Fun Listen!
My 12 year old daughter and I are huge Percy Jackson fans and this parody of the first book had us laughing. We loved his fart power and the changes in the personalities of the characters. There was even an unexpected twist at the end.
There were a few parts where the writing was a bit more crass than I like for my 12 year old. When I said I didn't like these parts, she of course rolled her eyes at me.
We would love more good quality parodies like these!

subset: commoncrawl


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Kad Kredit | 01 October 2017-31 December 2017
8x Reward Points + 'Priceless' Bali Trip for 2 on overseas spend in foreign currencies with your Hong Leong Mastercard Credit Cards
Campaign terms and conditions apply. Picture shown is for illustration purpose only.
* Applicable to selected Reward Points Mastercard Credit Cards.
** Total overseas spend with a minimum of RM1,000 for the month will be eligible.
None of Hong Leong Bank, ASEAN and Mastercard assume any responsibility for the products and services offered under this promotion. The products sold and services are provided solely by the relevant vendors, under such terms and conditions as determined by such vendors, and none of Hong Leong Bank, ASEAN or Mastercard accept any liability whatsoever in connection with such products and services. The products and services have not been certified by Hong Leong Bank, ASEAN or Mastercard and under no circumstances shall the inclusion of any product or service in this promotion be construed as an endorsement or recommendation of such product or service by Hong Leong Bank, ASEAN or Mastercard.


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Did anyone read Paul Sheehan's piece in the SMH today? I'm not quite sure what to make of it: The secret desires of men, and why they go unfulfilled:
We are awash with an appetite for romantic and sexual fantasy. Call it the Twilight phenomenon. It merely adds to the sexual suggestiveness which permeates our lives. But underline the word "fantasy".
Ooh, Sheehan attempts to be relevant with a clumsy Twilight reference. Which is particularly clumsy given the next few sentences are about divorce, that social trend commonly found in teenagers.
And then bingo – Sheehan is a cheer squad for Bettina Arndt, who is herself a cheer squad for old, privileged white men like Sheehan. Aww, isn't that sweet?
She is a one-woman battering ram against the suffocating excesses of feminist victimology, with its irritating assumptions of moral superiority.
You know, he's got a point here. I'm a feminist and I certainly feel morally superior to Sheehan.
He then spends a lot of time talking about erectile disfunction and older men who use Viagra. A lot of time. I'll leave it up to you to decide what that means.
Even by Sheehan's standards, this is a pretty rubbish opinion piece. I'm not sure if everything is my fault (because I'm a feminist), or if everything is the fault of men who don't accept that their own declining sexual interest is a part of nature, or if no one should ever smoke, or if people should be nicer to each other, or if he just wants to have Arndt's babies.


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Publication Date
The North Stirling Land Conservation District is located between Katanning and Albany on the northern edge of the Stirling Ranges in south-western Western Australia.It includes parts of the Shires of Cranbrook and Tambellup, covering approximately 100,000ha of the catchment of the North Stirling basin. The basin is a flat area formed on Eocene sediments with poorly defined drainage lines and many salt lakes. It is surrounded by low granite and sandstone hills. The climate is Mediterranean, with cool wet winters and hot dry summers. Average annual rainfall ranges from 375-500 mm per annum. There is very little surface or underground water flow through the basin and the area is subject to waterlogging and salinity owing to the rising water-tables following the clearing of native vegetation (Lewis1992). The Land Conservation District was formed in 1985 following concern about the rapid decline in productivity resulting from this salinity and waterlogging. There are about 80 landholders in the district, mostly involved in wool, barley, oat and lupin production.This manual has been produced to help you identify and map the soils on your farm. The manual has been compiled for the North Stirling Land Conservation District (L.C.D.) and a list of its members is included in Appendix 1. As well as assisting you to identify soil types, the manual encourages everyone within the L.C.D. to use the same terms so that individual soil maps can be compared.
Soil types, Soil management, Soil conservation, Land degradation, Water conservation, Soil samples, Crops, Pastures
Recommended Citation
Hardy, J, and Tille, P J. (1993), North Stirling Land Conservation District - soils manual. Department of Agriculture and Food, Western Australia, Perth. Report.
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Joseph The Silent
- Size: 5 x 7
- Pages: 186
- Item #:
- Our eBook fulfillment system has been designed for ease of use. Your eBook will be delivered to you securely in an .epub format, and will be watermarked with your name and information.
By Michel Gasnier, O.P.
The Gospels tell us almost nothing about St. Joseph, yet his life is full of spiritual treasures. Michel Gasnier O.P., here shows you where to find them and how they can enrich your own relationship with God. In this series of brief meditations he explores St. Joseph's work as a carpenter, his marriage, his character, the flight into Egypt, his return to Nazareth, Simeon's prophecy, and more. He gives you an enlightening portrait of this man who remains one of the Church's most extraordinary saints and intercessors.


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|Property type
|Minimum Stay
|1 - 7 nights
Location - Peninsula Grand is an oceanfront luxury condo complex located just north of town on Cozumel's west coast. The 12A condo is located on the south side of the building, offering unique, breathtaking views of the Caribbean, the town of San Miguel and the cruise ships!
Complex Amenities - Peninsula Grand has a large Infinity swimming pool, sandy beach and palapa area, snorkeling right off the beach, exercise room, elevators, and clubhouse.
Condo Features - Full kitchen, beautifully furnished, AC, washer/dryer, 24 hr security, satellite TV, spectacular ocean views from the living area and all bedrooms, safe and magic jack phone and internet.
Area Features - Cozumel is most famous for its world-class scuba diving, snorkeling and deep sea fishing. Water sports, jeep rentals, shopping and dining and rich culture will also add to your wonderful experience on this relaxing island.
Included in Rate - Towels/Linens, all utilities, housekeeping, purified bottled water, English/Spanish speaking guest services
This partner has demonstrated and is committed to fast response times, the best rates, and a great guest experience.
WOW doesn't begin to describe this condo. We were prepared to be impressed based upon the photos, but they really don't do this property justice. We communicated with Rentals Your Way about this unit and everything they told us about the unit and location was spot on. We really liked renting from this outfit due to their timely responses, professionalism and effective communication during the booking process. This was a fabulous condo with 3 private bdrooms with en suite bathrooms & a 4th bedroom that's great for kids. All of the furnishings were top quality. Kitchen outfitted for a queen/king who really likes to cook with high end appliances, cookware and dishware. Includes nice washer/dryer which helps reduce how much you need to pack. The bed and bath had high end linens. The bathroom towels coordinated with the bedroom themes which we really liked. It made this condo feel like a well loved home and not just a rental property.
Best of all was the view from the huge balcony. There's a very large balcony off the living area with nice couches/chairs and eating area and another very private balcony off the master bedroom. We were on the balcony extensively watching the sunset and one evening a nice lightning show over the ocean. You can hear the waves from the unit.
We rarely used the AC as there was such a nice breeze coming through the condo. At night we watched the huge TV in the living room which, surprisingly, included Netflix!! YEAH!
Pool was large heated infinity pool with plenty of space. Small hot tub was nice too. Below the pool is a beach area with sand and palapas that had power so you could charge your phone/tablet which was super convenient. Service was superior. The property manager we dealt with was Caribbean Beach Properties. Arrival and check in were so easy, a code was provided in advance. We liked having a code which means we didn't have to pack around a key while we were out. Caribbean Beach Properties also kept our room spotlessly clean. Other things we loved was the included snorkel/beach equipment in the laundry room. The onsite personal chef is a must do!!! He has a daily menu of things like fajitas, club sandwiches, guacamole, pico de gallo, margaritas and other items that were wonderful and extremely reasonably priced. Definitely use his services. It made us feel like an all inclusive because we didn't have to cook and had great food available all day! This was a classy place to stay!!
We have stayed at this complex many times and enjoy the property very much. The new cook is awesome. My kids enjoyed the heated pool and snorkeling in front of the condos.
The apartment was great! Very clean and nice. The view is amazing. Looks better in real than in the pictures. Everyone were so nice:). Recommend for sure and would totally come back.
Stayed for a week with 3 adults and 3 young children. The condo was great for our needs. One of the beds does not have privacy, which was not an issue for our group. Kitchen is quite functional. Views from the decks are fantastic. The pool was excellent for small children - plenty warm, with shallow areas for sitting/ wading and nice length for swimming. Decent snorkeling in front of the condo. Nice gym. Small beach - not a "walking beach" but again good for small kids. Perfect location north of town - very easy/ short drive to town for grocery shopping, restaurants, and access to dive shops. Enjoyed a tasty dinner cooked by the chef who works at the complex. Would definitely return.
Everything about this place was amazing. My only one complaint would be the hot tub by the pool was not heated. I will be back.
Condo was great! True to pictures and clean. Couple things, one of the beds is in the hallway so not much privacy and the jetted tub could be cleaner (stains in jets) overall great stay and beautiful view. Alex was awesome with helping us as well.
Cozumel is world-famous for its outstanding scuba diving and snorkeling. Glass bottom boat tours, deep-sea fishing, ATV and horseback riding adventures, Mayan ruins, and endless ocean activities are all available on the island.
National parks offer unbeatable swim with dolphin activities, snorkeling, sunbathing, and much, much more. Shop for jewelry, clothes, and souvenirs in Cozumel's quaint downtown area. Experience Cozumel's famous nightlife, whether you want a quiet, romantic dinner or a party atmosphere. Explore nearby islands or a take a quick 45 minute ferry ride to Playa del Carmen, Mexico's fastest growing resort destination.
Our professional reservations staff can answer any questions you may have regarding activities on the island or nearby destinations.
*Plus compulsory or optional charges based on actual occupancy or consumption. The payment has to be made in the currency offered by the property manager, before use of conversion tool. Payments in other currencies have to be negotiated beforehand with the property manager. The currency conversion tool is provided for your convenience only and is non-binding.


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CHICAGO — While writing his Nov. 5 column, ``Bush has drifted from mainstream,``
Clarence Page let the facts drift completely away from him.
It wasn`t the President who played a cruel ``pre-Halloween`` trick on the District of Columbia by vetoing its budget. It was anti-life zealots in Congress who deliberately crippled the bill with an abortion-expanding amendment.


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|Floor 13A
|3665 sqft
|RM7.50 /sqft
|Bare Unit
Floors N/A
Net Lettable Area (sqft) N/A
Building Class
Closest Public Transportation MRT Bukit Bintang
Internet Providers N/A
Pavilion Office Tower is located in the super prime area of Jalan Raja Chulan, annexed to the up market Pavilion Shopping Centre along Jalan Bukit Bintang. It is a 19 storey Grade A, corporate office tower with an average floor plate of 9,700 sq feet. The surrounding area is KL's shopping 'haven' because it is located within walking distance of Fahrenheit 88 Shopping Mall, Lot 10 Shopping Mall, Sungei Wang Plaza and Berjaya Times Square.
Pavilion Office Tower is also strategically situated at the entry point of the Elevated Walkway connecting KLCC and Jalan Bukit Bintang, which makes travelling between these 2 prime locations much more convenient.


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Celtic tattoos are one of the most ancient pictures on the human body, which can combine the deep meaning and the incredible beauty of the image itself. The explanation of Celtic tattoos is directly related to the religion of the northern people as well as to the beliefs of the Celtic mythology. Like many other nations they animated some animals and plants. But the historians are sure that Celts went further. – They were among the first ones in the world, who began to use symbolism in art. As a rule Celtic tattoos are abstract and involve a lot of secret signs.
Set Of Celtic Tattoo Designs
Tribal Celtic Tattoo Design
Awesome Celtic Tattoo on Back of Shoulder
Black and White Celtic Tattoo
Black and White Celtic Tattoo for Print on Arm
Honor Faith Unity – Celtic Cross Tattoo on Leg
Colourful Celtic Cross Tattoo on Arm
Small Celtic Tattoo on Body
Scary Celtic Lizard Tattoo on Back of Lady
Big Celtic Lizard Tattoo on Back of Man
Star with Moon Celtic Tattoo on Back
Scorpian Celtic Style Tattoo on Arm
Big Celtic Cross Tattoo on Back of Man
Nice Big Celtic Tattoo on Back of Body
Big Black Celtic Tattoo on Back
Big Celtic Cross Tattoo
Long and Big Celtic Tattoo on Back
Green Celtic Tattoo on Arm By Tattoobite
Coloured Celtic Tattoo on Arm
Big Celtic Tattoo on Bicep
Celtic Tattoo on Bicep
Celtic Cross Tattoo on Bicep by Tattoobite.com
Beautiful Celtic Tattoo on Bicep
Coloured Celtic Tattoo on Bicep
Tribal Celtic Tattoo on Bicep
Celtic Cross Tattoo on Bicep
Celtic Tree Tattoo on Back
Black and White Bird Celtic Tattoo
Celtic Zodiac Tattoo
Beautiful Celtic Tattoo
Black and White Celtic Tattoo for Shoulder
Maggy – Shamrock Celtic Style Tattoo
Nice Celtic Cross Tattoo
Celtic Viking Style Tattoo
Nice Celtic Armband Tattoo Design
Celtic Star Tattoo Design
Celtic Symbol Tattoo On Neck Back
Outline Celtic Tattoo Design
Cool Celtic Tattoo Design
Celtic Claddagh Tattoo On Upper Back
Wonderful Celtic Tattoo Photo
Full Sleeve Celtic Tattoo Design
Stunning Celtic Tattoo Designs
Celtic Tattoo On Back Shoulder
Celtic Armband Tattoo
Celtic Couple Band Tattoo
Celtic Knot Tattoo On Neck Back
Awesome Celtic Cross Tattoo Design
Tribal Celtic Tattoo Image
A Celtic Cross Tattoo On Biceps


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|Christopher Schultz <ch...@christopherschultz.net>
|Re: running tomcat6 under a different user than root (debian)
|Mon, 01 Nov 2010 14:38:03 GMT
-----BEGIN PGP SIGNED MESSAGE----- Hash: SHA1 Mark, On 10/29/2010 10:04 AM, Mark Thomas wrote: > On 29/10/2010 14:53, Ronald Klop wrote: >> If you have a webapp where users log in you can use there login/password >> to login on the database. A little bit inconvenient for the DBA but you >> don't have passwords on your servers. > > It isn't quite that clear cut. There are some trade-offs to make with > this approach (and I'm not sure I like them). > > 1. The user's password has to be available in plain text. That prevents > you from storing digested passwords in the realm. This can be avoided by using a random key created during webapp startup (and persisted across re-deploys, but not undeploy/deploy) to encrypt all the passwords during runtime. That key might still be discoverable using the techniques you've already described (reflection, etc.). - -chris -----BEGIN PGP SIGNATURE----- Version: GnuPG v1.4.10 (MingW32) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iEYEARECAAYFAkzO0MsACgkQ9CaO5/Lv0PAcqACgvWB5P4lsdOlIGwN8t4fY+S93 TgEAn2109aeRK0pGMAarSECByf1IiHS5 =101I -----END PGP SIGNATURE----- --------------------------------------------------------------------- To unsubscribe, e-mail: firstname.lastname@example.com For additional commands, e-mail: email@example.com
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Are You Taking Enough Risk to Enjoy a Comfortable Retirement?
Our latest research on Risk and Retirement was posted to the research section of our website. Below is a snippet from the piece. Click here to download the full report.
Our latest research on Risk and Retirement was posted to the research section of our website. Below is a snippet from the piece. Click here to download the full report.
The stock market enjoyed a remarkably quiet month of August as the S&P 500 finished flat and did not see a single day in which it moved more than 1%. In fact, on July 12th the S&P 500 was down 1.5% and has not seen a greater than 1% move since then. What a contrast to last August! This relative calm is reflected in VIX, the market fear gauge, though VIX only measures expected volatility 30 days out.
Longer term measures of volatility are higher than normal suggesting possible future volatility. In an election year, especially an election year with two unpopular major party nominees, this perhaps should be expected, though there are other explanations as well.
$VIX term structure today shows much less longer term complacency than other periods with low first future pic.twitter.com/SyHmiBWG5c— Eli Mintz (@VixCentral) August 15, 2016
Freshly carved out of the Financial sector, REITs are the newest Sector in the S&P 500. Sectors – like Financials, Information Technology and Consumer Staples – are the broadest classification of stocks with industry groups and sub-industries further demarcation points. The weighting of financial stocks in the S&P 500 fell by a fifth and REITs now comprise 3.1% of the index giving them a higher weight than materials, telecoms and utilities.
REITs were previously grouped with financials because they shared some qualities with financial stocks. Both Financials and REITs, for instance, have been more volatile than the S&P 500 as a whole with standard deviation of 7% compared to the index's standard deviation of 4%. But while mortgage REITs will continue to be classified as financial companies, the pairing with equity REITs made less sense as the correlation between eREITs and financial companies is less than the correlation between financials and the rest of the S&P 500.
Following the tumultuous aftermath of the Brexit vote in June investors were pleased to see the market deliver a strong month in July. The S&P 500 rose 3.7% and the Russell 2000 6%, verifying the trend we noticed in mid-July, as stocks largely outperformed second quarter earnings estimates. The S&P 500 is now up 7.7% year-to-date and the Russell 2000 8.3%.
Most widely-used stock indices you see, including the S&P 500, are weighted by market capitalization rather than weighted equally, giving the larger stocks in the index more sway over the index's movement. The 10 largest stocks by capitalization in the S&P 500, for instance, have the same weighting as 89 equally weighted stocks.
Because market-cap weighted indexes are heavily biased by the largest constituents, these indexes typically publish a less followed but insightful Equal Weight Index (EWI) that weighs each stock equally across the index. Apple makes up 2.9% of the S&P 500 Index but just 0.2% in the S&P 500 EWI.
By removing the extra influence large-caps have in the standard index, we are able to pick up on trends by comparing the performance of both the market-cap and the EWI version to see how smaller-cap constituents are performing relative to their larger peers.
The recent returns of the S&P 500 indices show signs of size movement. The large-cap S&P 500 is no longer outperforming the small-cap S&P 600 as it did in the prior 1-year period. Mid-caps, as seen by the S&P 400, are now returning more than both small and large caps. And, interestingly, within each index, the EWI version outperformed the cap-weighted index indicating, at least in 2016, small-caps have taken the momentum from large-cap stocks.


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WYCKOFF, N.J. -- Daniel P. Brovero, 76, of Farmingdale, died Wednesday, July 27, 2016.
Born in Englewood, he lived in Wyckoff for 42 years before moving to Farmingdale two years ago.
Before his retirement in 1996, he owned and operated the Forest Café in Englewood for more than 30 years.
He was a former parishioner of St. Elizabeth's RC Church in Wyckoff.
Surviving is his wife of 52 years, Donna (nee Swenson) Brovero; children, Donald and his wife Margaret of Monroeville, Danielle Sheehy and her husband Michael of Buckingham, Pa. and Debra Murphy and her husband Stephen of Westfield. Also surviving is his sister, Pauline Cresanti of Emerson; and eight grandchildren, Dakota, Christopher, Jarrod, Sarah, Colin, Justice, Griffin and Sienna.
Visiting hours are from 2-5 p.m. Sunday at the Vander Plaat Funeral Home, 257 Godwin Ave., Wyckoff. There will be a funeral Mass at 10 a.m. Monday at St. Elizabeth's RC Church, 700 Wyckoff Ave., Wyckoff. Interment to follow at George Washington Memorial Park in Paramus.
In lieu of flowers, please make donations in his honor to Leukemia Fighters/NY Presbyterian Hospital, c/o Dr. Gail J. Roboz, 520 E. 70th St., P.O. Box 403, N.Y., NY 10021.
Click here to sign up for Daily Voice's free daily emails and news alerts.


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The color scheme makes or breaks an interior design. Rustic kitchen signature color schemes are comprised of shades of red, orange and gold. Orange color is said to be the appetite stimulant and comfort maker. The floor should be distinguished with hard wood and metallic copper shades to give it a Mediterranean feel. Wall painting must illustrate the rustic design feeling.Home owners can try orange, red, shades of gold, olive green and darkest shades of Tuscan style furniture.
How does one design with a country theme kitchen? One of the most important factors is the colors used in the kitchen. Those that bring in warmth and a fresh and clean look and feel are often recommended. Softer hues of green, yellow and brown may be preferred as well as other earthly colors. To help achieve this effect, kitchen owners will often flower and plant details into the area through wallpapers, frames or the real thins in vases and pots.
Home owners can apply excellent interior design ideas to their kitchens. Kitchens can be given a more retro or a completely utopian design, whichever suits the owner's personality and preferences. Every home owner wants their kitchen to look like the one they see in the interior design magazines and templates. That dream is not difficult to achieve. A home owner can even have a kitchen that looks better than pictured demos with careful planning.
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Many of our clients are relieved to find that acupuncture for migraine headaches is a viable alternative to the current crop of pharmaceutical/medical migraine interventions.
According to traditional Chinese medicine, pain of any type is synonymous with a blockage of qi, or the body's vital energy. This qi blockage can be caused by a number of different factors, which in the case of migraine headaches usually fall into a few main types. These include stagnation of blood, often due to trauma or injury even decades old; disharmony of the liver and/or gallbladder channels, or an imbalance in (usually) female hormones. Fortunately for the consumer of acupuncture, it has been our experience that acupuncture for the treatment of migraine headaches is usually very effective.
What can one expect if coming in for acupuncture for migraines? Often, patients report a reduction of pain within seconds of the insertion of the needles. Certainly by the time needles are removed one half to one hour later, most patients find their symptoms noticeably improved. This can include less pain, lessening of visual disturbances, a decrease in nausea, and less over-sensitivity to light and sound.
Sometimes, acupuncture for migraine headaches provides a quick fix, and patients are finished with treatment after only a handful of sessions. Much more commonly, however, is the case where the pattern of disharmony leading to migraine headaches is firmly entrenched, and a longer course of treatment is indicated. This might look like acupuncture sessions 2 to 3 times per week for several months before good results are lasting.
Many times, we will recommend the use of Chinese herbal formulae, specifically tailored to each person's exact pattern of disharmony, to strengthen and consolidate the effects gained by using acupuncture for migraine headaches. To learn more about Chinese herbology, the herbs we use, and how they are prescribed, click here.
To recap, acupuncture is often received as a God sendť by those suffering from acute or chronic occurrences of migraine headaches. Consistent treatment with acupuncture almost always results in a decrease in the severity and in the frequency of migraine attacks. If you need help with migraines or feel like you are out of options with your current treatment plan, consider receiving acupuncture for migraines and see why so many relieved patients recommend it.
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Acupuncture in Ashland, Oregon - Serving Southern Oregon, the Rogue Valley, Medford, Ashland | SiteMap
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Meringue Edging Crochet Blanket Border – Tutorial
- Yarn recommendation – For this tutorial, you can use a soft, sturdy economical powerhouse like KnitPicks Brava Sport (Affiliate). See a full review of this yarn on my crafty site.
- Hook Recommendation – I own and use the hook from this set on Amazon (Affiliate)
Meringue Edging Crochet Blanket Border Tutorial
Pssst! Looking for more crochet blanket border patterns? Check it out!
This edging can be used on a straight DC or a granny edge as in this project. If you're doing a straight DC border, just skip 3 stitches between the shell and the SC like if it were a granny.
I work a round of the border color first, so when I start, I'm not attaching new yarn. I finish the round in a chain 2 corner. Essentially the shell is: 3DC, Picot, 4DC
*See how to make the Picot below*
Edging: (SC, chain 1, 3DC, Picot, 6DC) in chain 2 corner (Corner shell made). *SC in the next ch2 space. (3DC, Picot, 4DC) in next ch2 sp* repeat across to the next corner.
At my corners, I come to a shell at the end, when I should be SC-ing before the corner. I just play it off and do the corner shell anyway! It makes for a great leafy corner and I love it as an added piece of the design.
After you 3DC, chain 3
Insert your hook in the DC, through the center with the two outer bars on the outside, like in the photo below.
Yarn over and pull the loop through to make an SC.
Finish the SC. Picot made.
So, chain 3, SC in base stitch. This base stitch could be an SC too, if you're just working SC across with picots at an interval. By the way, that makes a pretty, delicate edge.
Have fun, and let me know how it goes!
Categories: Free Patterns


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EAL Session:
"I was thrilled to be invited for an EAL session with Aimee, and as a lover of horses, professional development and new experiences, I couldn't wait to join her on her beautiful property. I was aware of EAL through previous work and study, but this was the first time that I had been the recipient of a session. The experience was incredibly soulful, grounding and heart-warming for me.
Aimee's enriching, supportive and gentle guidance and Takoda and Poppy's (the horses) willingness to allow me to explore myself and my abilities in a new (and sometimes challenging!) way provided the perfect environment for me to reconnect with important life lessons and openly reflect on what I most want for my time ahead. Aimee is fantastic at what she does and my experience with her will stay in my mind for a very long time to come."
Johanna Parker, Life Coach at Heart Sparks
Life coaching series:
"When I came to Aimee I was firmly in procrastination station. The vision for my life and my career had really started forming, but I just couldn't pass through the blocks that were keeping me from action. With Aimee's loving, intuitive and humorous support, I was able to sift through the multiple things that were pulling at my attention and connect with what truly mattered to me.
Thanks to Aimee's practical and spiritual support (including some loving 'kicks up the butt'), I finally gained the confidence and clarity to publish my coaching website and have a business vision for the future that exactly reflects my vision and my current personal journey.
If I had to describe my coaching experience in just three words? Transformative, aligning and a whole lot of fun"
Lucy Taylor, Webdesigner for wellness entrepreneurs at Truly You Websites
EAL session:
"Connecting with the horses was just beautiful and such an incredible experience. I've ridden horses before but this was different, it actually made me quite emotional. Feeling the textures of the horses and patting them, feeling the sun against their backs and how they responded to my touch was very calming. The process took me back to nature and my childhood, which is a time I always remember fondly. It made me realise how constricted I'd been feeling and how getting out in nature and also getting my hands dirty is a way I can start feeling more relaxed and free.
I hadn't anticipated just how much I needed this, and how much of an impact the horses would make. Aimee was an incredible guide and her follow up session helped me identify even deeper insights."
Katie Maynes, PR CONSULTANT FOR ENTREPRENEURS at Katie Maynes
Life coaching series:
"I was finding some things a little overwhelming in terms of what am I going to do with my life. I hadn't worked with a coach before and I wasn't sure what to expect.
Coaching with Aimee has re-established what is important for me at this stage of my life to keep me feeling balanced and in control of my feelings and emotions. It has re-established my priorities. I now have the confidence to follow my passion of writing in way that looks and feels right to me.
I always felt that Aimee was 100% dedicated to helping me achieve my goals and to helping me become the best version of myself that I could be. Her enthusiasm and encouragement was contagious and I always came away from our sessions feeling excited and focused."
Melissa Millard, Writer
EAL Session:
"Out of curiosity I decided to try a one-off EAL session with Aimee. I hadn't been around horses in years but thought it would be interesting to try and see what came from it. It was one of the best decisions I have ever made. I was a bit nervous around the horses at the start but Aimee quickly put me at ease.
Throughout the session I felt cared for, attended to but also allowed to be in my own space and completely present with the experience. Upon reflection, two parts of the session really stood out for me. The first was feeling so connected to the horses and being able to just be in their physical presence. This was so powerfully grounding for me. The second was the afterglow. I was on a high for the rest of the day – and the next day too! It was really amazing to experience something that resonated so deeply with me.
The whole experience was so incredibly grounding, powerful and special that I can't wait to return for more. If you even have an inkling that you would like to go and experience an EAL session with Aimee, I highly recommend it. Book in now!"
Jade McKenzie, Event professional and coach at Event Head
Life coaching series:
"Aimee came into my life when I was feeling quite disconnected, stuck and confused. A gifted Intuitive, she knew exactly how to cut through the BS and limiting beliefs that had been holding me hostage.
Aimee helped me to set inspiring goals that were aligned with who I am and where I wanted to be. A natural encourager, she helped shine a light on areas where I wasn't being true to myself...but also gave me a gentle ass kicking when I needed it most!
After my coaching series with Aimee I felt truly nourished, connected and balanced. I can't thank her enough for helping me see the light again."
Jade Ogden, Wellness blogger at Make Your Life Amazing
EAL Session
"My experience with Aimee and the beautiful horses Takoda and Poppy was incredibly grounding, and I felt completely present and held by nature. Not only was I able to uncover some learnings that had been continually popping up for me, but I also gained clarity around the best path forward for me in these areas. It was a completely new experience for me and one that I would recommend to anyone who feels called. I can't speak highly enough of this wonderful session!"
Abby Lewtas, Life Coach & Speaker and Abbylewtas.com
Life coaching series:
"I gained so much from my coaching sessions with Aimee. Her passion for Life Coaching and helping people was evident to me every session. It was the first time someone had held me accountable for my actions when it came to my business.
As a result of learning how to be more mindful strategic and considered, I was able to achieve a significant promotion in my company. I am confident that I would not have achieved this success without her support, humour and encouragement. Aimee gave me some incrediby useful ideas, helped me set goals, and introduced me to some resources that I have since been able to integrate into my business.
Aimee believed in me possibly more than I believed in myself throughout the coaching sessions. I have no doubt Aimee will do well in this profession, and change the lives of many more lucky people!"
Paula Cook, Independent Presenter for Younique at Paula Cook Makeup
EAL Session
"I did an EAL session with Aimee and at first I was a little unsure that I would get much out of it, I guess you could say I was almost skeptical. But once I had my introduction with her beautiful horses, I instantly felt an energy from Poppy – it was as if she was an old soul, wise and caring. Until I experienced that for myself I wouldn't have believed you could connect with an animal in that way.
We did a short meditation exercise. Aimee was a fantastic guide, she kept it to a level that suited a beginner like me, made me feel extremely comfortable and at ease. Her clear instructions and calming tone made for a relaxing meditation and encouraged a realisation of the different sensations I experienced in my body that I normally would not be aware of.
Aimee was able to use these realisations as the focal point of our session, guiding me to connect with Takoda and observe how he responded to that energy. It was a surreal experience that was essentially magical! Aimee has the skills and facilities to provide something truly unique. I will definitely be going back and recommending her to everyone I meet!"


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Experiences with Treatment after surgery experiences
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- Experience with Breast Cancer
Treatment after surgery
Decision Point
will know more after pathology results thursday
- gisellecp
- Experience with Ovarian and Fallopia...
Treatment after surgery
Decision Point
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An international team of researchers released a study last week concluded vaping is good for public health. The study revealed that for people born in 1997 the rate of death from smoking will fall by 21% thanks to electronic cigarettes. The study was published in the Oxford Journals Nicotine And Tobacco Research. The study was funded by the National Institute on Drug Abuse, the National Cancer Institute and the Cancer Intervention and Surveillance Monitoring Network.
The study relied on modeling using a number of calculations of modified risk of electronic cigarettes vs tobacco. The models ran scenarios ranging from 25% of the risk of smoking down to 2.5 % of the risk of smoking. Regarding the question of 'is nicotine bad for you' scientists recognize that nicotine is not what contributes to smoking deaths but nicotine exposure to people under 21 is still a concern.
Medical Professionals Concluded Vaping Is Good For Public Health
Dr. Laura Crotty-Alexander, UC San Diego assistant clinical professor of medicine as well as a staff physician at VA San Diego Healthcare System, said that this study shows the possible life saving possibilities of electronic cigarettes. Dr. Crotty-Alexander said, "This article shines a light on the potentially dramatic and beneficial consequences of nicotine delivery devices (NDDs) in the U.S."
Researchers cautioned against oppressive regulation that could limit the availability of quality electronic cigarette products that could further enhance the opportunity for smokers to access a less harmful alternative. They also cautioned against government using misleading information that could potentially keep smokers away from vaping. Study leader David Levy said that government plays a role and when misleading statements are made about vaping it leads to misunderstanding and mistrust in government.
Many experts agree that regulation should play a role and focus on product safety as well as quality standards. Unfortunately, if the current FDA ecig regulations go forward as planned smokers will pay the heaviest price.


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UCR pre-pharmacy
Think about what career is best suited for you, and where you want to study. Request more information now.
Think about what career is best suited for you, and where you want to study. Request more information now.
Think about what career is best suited for you, and where you want to study. Request more information now.
Information about UCR pre-pharmacy, registering for degree programs, and taking free tests online. Nursing is one of the fastest-growing job areas, and for good reason. As the population ages, medical care will continue to expand. If you study pharmacy, for example, there are a variety of concentrations that may appeal to you such as pharmacist assistant jobs, pharmaceutical company internships, or drug rehabilitation. Check with the pharmacy department for an updated schedule of classes, registration deadlines, and a list of pre-pharmacy degree requirements. Due to advances in health care, there is a growing skill-shortage of pharmacists, and of qualified medical personnel in general. Please use the form above to request admissions info for UCR.
Healthcare jobs such as pharmacy technicians, LPN's, and LVN's provide over 15 million jobs in the United States alone. Most healthcare jobs require 4 years of college, such as a pre-pharmacy degree, or bachelors degree in biology. The American Hospital Association is the national organization that represents and serves all types of hospitals, health care networks, and their patients and communities. Founded in 1898, the American Hospital Association provides education for health care leaders and is a source of information on pharmacist assistant internships and public health programs.
The outlook for pharmacy careers is especially bright. Employment opportunities for pharmacy technicians, for example, are expected to increase by 15% over the next ten years. The increasing number of elderly people is seen as a factor in this growth. As people age they have more medical problems, and hospitals will require more pharmacists to work in the dispensary. Aside from a good salary, most healthcare personnel receive comprehensive medical and dental benefits, as well as retirement plans.
REGISTERED NURSE, HOSPITAL (OR, Per-Diem)
UMass Memorial Health Care
Participates in and supports Nursing Governance activities. Bachelor of Science degree in Nursing highly preferred..
1 hour ago Registered Nurse (RN)- Behavioral Health- East
Eastside Medical Center - Snellville
Nursing Plan of Care is developed to meet individual patient needs according to nursing standards of care/practice within 24 hours of admissions.
4 hours ago SMU-Full Time FNP Nursing Faculty (Sacramento Campus)
Sutter Health
RN Licensure and California Board of Registered Nursing approved APRN. Faculty is responsible for creating environments that facilitate student learning and..
3 hours ago Case Management Manager
Meridy Staffing Solutions
Bachelor's degree in Nursing or equivalent experience. Thorough knowledge of case and/or utilization management and clinical nursing..
13 hours ago Travel Registered Nurse Labor & Delivery (L&D) / OB
TravelMax Nursing
Benefits for our Travel / Contract Assignments Include: Varying Length Contracts with Premium Compensation Packages Medical, dental and vision insurance 401
20 hours ago Swim Teacher - Part Time and Summer Positions
Emler Swim School
Our teachers come from a variety of backgrounds, including coaching, retail, teaching, kinesiology, child development, early education, youth sports leadership..
3 hours ago


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Although it seems like a simple concept, the role of protein in our bodies and as a nutrient is often misunderstood. There is some debate today on whether North Americans consume too much or too little protein. On one hand, we always hear that we should decrease the amount of meat we eat, and that maybe a vegetarian diet is the best solution for a lot of the common health concerns seen today. On the other hand, there are certain weight loss diets that claim that high protein diets are the best way to lose weight. So who do we believe?
Protein requirements for an individual are calculated to be about 0.8 grams of protein per kilogram of body weight. That means that someone who is 140 lbs (approximately 63.5 kg) should eat about 51 grams of protein per day. For athletes, growing kids, or others who expend more energy on a daily basis, the requirements can increase to 1-1.2g of protein per kilogram of body weight.
Why We Need Protein:
- Proteins are a part of many different parts of our body—skin, muscles, organs, blood, etc. They are also found in enzymes, immune molecules, and hormones.
- It is important in energy production, muscle contraction, and overall growth and development.
Some symptoms of protein deficiency include:
- Weight loss (and inability to lose weight)
- Fatigue and lethargy
- Headaches
- Poor wound healing (bruises, cuts, etc)
- Recurrent or nagging sports-related injuries
- Thinning or brittle hair and nails
- Protein deficiency has also been associated with symptoms of depression, anxiety, and mood changes.
- Eggs: 1 whole egg contains 6 grams of protein; 1 egg white contains 4g of protein and is really one of the few pure protein sources available (1 egg white contains only 16 calories while a whole egg has about 90)
- Chicken breast (skinless): 4 oz contains 35 grams of protein
- Turkey breast (skinless): 4 oz contains 34 grams of protein
- Salmon: 4 oz contains about 30 grams of protein
- Halibut: 4 oz contains about 30 grams of protein
Vegetarian Sources of Protein:
- Quinoa: 1 cup of cooked quinoa contains 8 grams of protein
- Almonds: 23 almonds contains 6 grams of protein
- Tofu: ½ cup has 8 grams of protein
- Tempeh: ½ cup has 15 grams of protein
- Beans such as chick peas, kidney beans, and lentils: 7-9 grams per ½ cup
- Protein can also be found in smaller proportions in other foods that contain grains such as bread, pasta, and rice.
All the best,
Images from www.corbisimages.com
Dr. Cho, ND is a naturopathic doctor based in Pickering, Ontario and Richmond Hill, Ontario. She maintains a private practice focused in pain management and sports nutrition, in addition to a general family practice at Durham Natural Health Centre. To learn more about Dr. Cho, book a complimentary 15 minute consult by clicking here. Not in the Durham Region? Contact her through firstname.lastname@example.com to learn about more options.


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Hi, I would like to have some info about Boracay.
I want to go there next winter, maybe in January.
I heard it's nice but crowded.
Someone has been there this winter?
How is the "pollution" situation?
Thanks in advance
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You are an inspiration to many students and you have influenced many teachers in the field. Keep up the good work and never quit doing what you are purposed to do. I love you so much.


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Locksmiths in Sorrento offer a highly demanded service, which generally relates to maintaining and installing the various types of lock systems, from the standard key locks to the complex electronic or biometric locks. The most common types of locksmith professions consist of the emergency, industrial, commercial, and residential, which each of the specific fields requiring different skills and abilities.
Residential work is likely to be the most common of the services provided by a residential locksmith. A domestic property owner looks at personal security and safety as a key reason when it comes to making sure a home is fully secured against a would-be intruder. A locksmith has gained the experience and knowledge to suggest the most effective locks for a properties doors and windows. Beyond the ability to install new locking systems, the locksmith is also able to repair or replace the locks on the older styled properties. Extra services offered by a local locksmiths offer security advice, a key cutting service, installing locks on a garage or similar outbuilding, and installing at-home safes or vaults.
How Do I Choose A Residential Locksmith in Sorrento?
Nobody thinks that they will need the services of an emergency locksmith but regrettably things can occur that are just out of our control and you can find yourself unexpectedly needing the services of a 24 hour emergency locksmith to get you securely into your home, office, warehouse or even vehicle.
Locksmiths services may not just cover common lock picking and gaining entry into a buildings, services may also encompass other security associated services together with vehicle security, emergency boarding up, burglar alarm fitting and maintenance, CCTV fitting and maintenance and even more specialist services such as Door entry systems and building access control.
Vehicle Security and automotive locksmith services
Some locksmiths may also offer automotive locksmith services and may be able to gain access to your vehicle if you misplace your car keys or lock them in your vehicle. The objective of an automotive locksmith is to gain access to your vehicle without damaging the pricey locks which are more often than not more costly to replace than basic door and window locks.
An automotive locksmith may also offer key cutting and professional key coding allowing you to have spare electronic keys for your vehicle, these services typically cost much less than they would if you were to go straight to the vehicle manufacturer.
Emergency Boarding Up
Not all locksmiths offer emergency 24 hour boarding up services due to the fact that 99% of these jobs take do place at night or in the early hours of the morning, those that do may insist on a call out fee because of the anti-social hour on top of the boarding up fee which may depend on the dimensions of the window and the damage. It's very unusual to see a locksmith offering a fixed price boarding up service.
Boarding up is just a short-term measure to protect your home, office or business premises until the next day when your window or door can be appropriately replaced. When sourcing a locksmith or security company for boarding up services it is always best to source someone who is local and independent rather than using a nationwide call centre, this way you can be confident that you are speaking direct to the locksmith and get precise arrival times, after all you don't want to be waiting all night.
These are just some of the services that you can expect from a locksmith or security firm, 247 Safeguard Locksmiths are a Emergency Locksmith Peterborough based professional locksmith company who strive to deliver competitively priced locksmith and security services to both domestic and commercial clients throughout Lincolnshire and Nottinghamshire.
Car Locksmiths
We all know the uses of locks: they provide security for our vehicles, enterprises and houses. But when you lock yourself out of your house, you wish locks didn't exist. In such a situation, you need the services of a good locksmith. The locksmith, however, should be reliable and trustworthy. Given below are some tips that can help you find a good one.
Hire a local locksmith
If you hire a locksmith located far away from your residence, you should be ready to pay a bit more money. The reason is that the service charges will also include the traveling expenses. Generally, the offices of local locksmiths can be visited any time. We suggest that you prefer a local locksmith.
Get referrals
You may ask your friends and family for referrals. If you live in a village or town, you may not find more than a few locksmiths in your area. However, in big towns, you may have a lot more professionals to choose from. This will help you choose the best professional.
Get ready
Looking for a reliable locksmith is not as hard as you may think. However, you should be ready to deal with a crisis. You may want to have a contact list of good professionals in your phone at all times. Alternatively, you can also keep their contact details in a diary.
So, if you have been looking for a good locksmith to meet your needs, we suggest that you don't hire any professional that you may know. Instead, you may want to consider the tips given in this article before making your choice.


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Morrowind is the latest addition to The Elder Scrolls Online series and it's out now. Join over 10 million players worldwide in the game that keeps getting bigger. With hundreds of hours of base-game content, five released DLC game packs, the new Morrowind Chapter, and upcoming Horns of the Reach and Clockwork City DLC game […]
Posts Tagged 'Morrowind'
Morrowind: The Elder Scrolls Online – E3 2017 Trailer
Posted by Dom Robinson on June 13th, 2017


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Bubblegum Junior is a fun range of soft seating, available in a great choice of bubblegum colours. The modular design offers maximum flexibility and with matching accessories, you can configure Bubblegum Junior to fit your space and budget.
Furniture in its most flattering form. Take a look through our gallery and see how our products integrate and interact with each other
Come and see our products in action; here you'll find our most recent installations for you to browse.
Send us your query now and we will get back to you.


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Fitness exercise bikes are great for low impact workouts. Recumbent exercise bicycles are especially beneficial, and on these models the rider sits in a bucket seat leaning slightly backward. This is a more comfortable position for those who are overweight or have back problems.
Greater Physical Benefits
Since using a recumbent exercise bike is more ergonomically correct, the abdominal muscles relax, making breathing deeper and easier (and thereby improving the cardiovascular workout). This in turn increases the aerobic capacity and also the burning of fat. Recumbent bikes not only exercise the gluteal muscles, but shape the butt area as well. There are no muscle strains on the back, shoulders, neck, ankles, or knees since the rider is not hunched over.
Recumbent Bikes Have More Features
Recumbent bikes have a number of different settings to increase resistance, which produces a great fat-burning cardio workout and builds muscles. Simulating different types of courses from easy and flat to difficult and hilly, they also come with:
- CD displays
- heart rate monitor
- preset programs
- other informational systems
The Schwinn exercise bicycles 230 and 231 range from $460 to $620 and have 12 to 23 programs. The G-force RT Lemond fitness bike has 13 different programs and costs approximately $2,600. The Vision Fitness Exercise Bike is semi-recumbent in that its seat rides higher than the pedals. All of these are great choices to get you on a successful (and low impact) path to fitness!


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Another Great Hinterland Sale
This beautifully presented rendered brick and tile home is sure to impress. Offering three bedrooms all with built in robes and ceiling fans, updated bathroom and kitchen with glass splash back, stainless steel dishwasher and range hood, gas cooking, heating and hot water. Timber floating floors through out, reverse cycle air conditioning and back to base alarm system & a single carport.
Sitting from the lounge room you can look out the timber french doors that lead out to a large timber deck running across the front of the home giving you a great view of Gosford CBD and Showground, so sit back on new years eve and relax watching the fireworks from your own balcony.
All this on a 683m2 block, minutes from train stations, shopping centres and Schools this home is a must to inspect.
Property Features
- 3 bed
- 1 bath
- 1 Parking Spaces
- Land is 683 sqm
- Carport
- Sold
- Satellite
- Street View
- Transit
- Bike
- Comparables


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978-291-0002 (o))
Frank Liquori has been selling alot of cues to APA members. His work has a good reputation among the APAplayers, many who have purchased custom cues from him. He makes a shaft that isamazing. APA members are offered a 10% discount. The company name is Liquori Billiard Products and the number is781-763-7776. Call Frank to see his cues in person.Frank just got a new shipment of exotic woods. Frank makes his cues mostly fromexotic woods. However, he also makes some inexpensive cues from woods such asMahogany.
A new APA member discount is now avaliable at Earth Creations Get a 10% discount on all supplies they sell. Discounts on loam/mulch crushed stone pavers everything They sell.Earth Creations 12 kidder Rd Chelmsford MA 01824 978-674-8434 -- Special thanks to owner Dennis Garneau Jr
APA members: Does your children's park or school need a new affordable playground or could your business benefit from a high quality custom lettered bench with your logo?
Fellow APA member Steve Dibble (APA rank 6 plays at Salem Moose) owns DibblePARKS and is happy to offer a 10% discount on all products to APA members in New England. DibblePARKS has designed and built over 900 public playgrounds throughout all of New England. Reference your APA membership # and get your children 10% discount off a new playground for their school. DibblePARKS also carries a full line of Webcoat metal benches and picnic tables; sit-in, sit-on, and stand on kayaks: bleachers and sports equipment (soccer & lacrosse goals etc); 3 season shade structures and permanent gazebos of all types; and lots more park and gym related equipment – all will get 10% APA Discount. Full catalogs can be seen on their website www.Dibbleparks.com or call DibblePARKS at 978 744 7815.
Steve Dibble
74 Moffatt Road
Salem, MA 01970
Direct: (603) 770-3380
Office (978) 744-7815
Fax (978) 744-1388
Email: email@example.com
Website: www.DibblePARKS.com
Special offer: If a current APA player contacts Mike directly at 888-MEAHUNA or 888-632-4862 shipping is FREE. Order before 10:00 pm est on Tuesday, December 20th and delivery will be guaranteed by December 23rd. They also donate $1per pound towards a charitable organization. Fundraisers for schools, organizations and teams are available. www.kopekapeallc.com or www.facebook.com/kopekapeallc
Thanks Mike!